CN112522143A - Novel kitchen waste high-temperature composting process assisted by complex enzyme preparation and ochrobactrum anthropi - Google Patents

Novel kitchen waste high-temperature composting process assisted by complex enzyme preparation and ochrobactrum anthropi Download PDF

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CN112522143A
CN112522143A CN202011417785.4A CN202011417785A CN112522143A CN 112522143 A CN112522143 A CN 112522143A CN 202011417785 A CN202011417785 A CN 202011417785A CN 112522143 A CN112522143 A CN 112522143A
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temperature
ochrobactrum
composting
total mass
materials
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方光玉
徐有新
张文武
周华文
范一
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Shaanxi Xilixiuchuan Biotechnology Partnership LP
Yulin Heyi Aerospace Technology Innovation Co ltd
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Shaanxi Xilixiuchuan Biotechnology Partnership LP
Yulin Heyi Aerospace Technology Innovation Co ltd
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09B—DISPOSAL OF SOLID WASTE
    • B09B1/00—Dumping solid waste
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05D—INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
    • C05D3/00—Calcareous fertilisers
    • C05D3/02—Calcareous fertilisers from limestone, calcium carbonate, calcium hydrate, slaked lime, calcium oxide, waste calcium products
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
    • C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
    • C05F17/50—Treatments combining two or more different biological or biochemical treatments, e.g. anaerobic and aerobic treatment or vermicomposting and aerobic treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01004—Phospholipase A2 (3.1.1.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01005—Lysophospholipase (3.1.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21064—Peptidase K (3.4.21.64)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
    • Y02W30/00—Technologies for solid waste management
    • Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse

Abstract

The invention relates to the field of microbial technology and environmental engineering, and discloses a novel kitchen waste high-temperature composting process assisted by a complex enzyme preparation and ochrobactrum. The preservation number of the Ochrobactrum anthropi is CGMCC No.19230, and the microorganism is named as Ochrobactrum sp. The complex enzyme preparation and the bacterial strain of the invention can be used for high-temperature aerobic composting treatment of various organic matters such as kitchen garbage, agricultural product tailings, slaughterhouse wastes and the like by matching with a high-temperature composting process, so that the organic matters are completely humified, dehydrated and dried, and reduced by 70-92%, and finally the organic fertilizer with excellent quality is formed. The novel high-temperature composting process also relates to special pretreatment means such as physical cutting, enzymatic hydrolysis, rapid heat generation of bacterial strain metabolism and the like, so that the compost can be rapidly heated to above 70 ℃ within 8 hours, the high-temperature stage of composting is rapidly entered, various high-temperature strains are rapidly changed into absolute dominant strains in a compost system, and on the basis of the absolute dominant strains, the semi-continuous composting fermentation process capable of feeding and discharging materials every day is realized.

Description

Novel kitchen waste high-temperature composting process assisted by complex enzyme preparation and ochrobactrum anthropi
Technical Field
The invention relates to the field of microbial technology and environmental engineering, in particular to a novel kitchen waste high-temperature composting process assisted by a complex enzyme preparation and ochrobactrum.
Background
With the vigorous development of the economy of China, the living standard of people is continuously improved, the yield of domestic kitchen garbage rises year by year, and the annual yield of domestic kitchen garbage at present exceeds 1 hundred million tons. The kitchen waste contains a large amount of organic matters including oil, cellulose, saccharides, proteins and the like. If the treatment is not scientific and reasonable, the kitchen waste not only can produce solid pollution, but also can produce serious wastewater pollution and stink pollution after being rotten, so that the living environment is seriously damaged, and the life of residents is influenced. In recent years, the high-temperature composting technology developed by utilizing high-temperature resistant microorganisms can effectively treat kitchen garbage, so that the kitchen garbage is highly humified to generate organic fertilizer capable of being recycled. High temperature composting is a process of decomposing organic matter into humus by metabolism of heat-resistant microorganisms under high temperature conditions. High-temperature composting can be classified into aerobic high-temperature composting and anaerobic high-temperature composting according to the type of respiration of microorganisms used. At present, the aerobic high-temperature composting technology is widely applied at home and abroad. Compared with the anaerobic high-temperature composting technology, the aerobic high-temperature composting has the following advantages: firstly, the malodorous substances generated by aerobic high-temperature composting are few, and the bad odor substances have no influence on the air basically. And secondly, the degradation rate of the aerobic high-temperature compost on organic matters is far greater than that of the anaerobic high-temperature compost, namely the degradation rate of the aerobic high-temperature compost is higher. And thirdly, the aerobic high-temperature compost is suitable for small chemical fertilizer piles and can be applied to distributed garbage treatment, and the anaerobic condition is ensured without large-scale fertilizer piles.
The fermentation temperature of the traditional compost is generally not more than 60 ℃, the reaction time can last for about 15 days, the types of microorganisms participating in the composting process are various, and the microorganisms also comprise a plurality of strains which can generate malodorous substances such as cadaverine, putrescine and the like, so that the traditional compost fermentation can generate unpleasant peculiar smell. The high temperature compost fermentation temperature is higher than 70 ℃, conventional microorganisms cannot survive, and special high temperature resistant microorganisms such as Pseudomonas taiwanensis and Chelativorans composii need to be used. Because the species of the microorganisms which grow and propagate are only limited to high-temperature resistant strains under the high-temperature condition, the strains which produce the odor die, and the odor substances can not be produced in the fermentation process. And the fermentation speed is accelerated under the high-temperature condition, and the water evaporation is faster, so the speed and the quality of producing the organic fertilizer by high-temperature composting are higher.
Although high-temperature composting has a plurality of advantages, in the practical engineering application process, because the propagation rate of high-temperature resistant microorganisms at normal temperature is very slow and even can not be propagated, the temperature of the fertilizer pile is always ensured to be in the ecological advantage of high-temperature resistant strains by electrically assisting in heating, and the energy consumption is additionally increased. In addition, the substrate spectrum of the high-temperature resistant strains commonly used at present is narrow, most of the high-temperature resistant strains can only directly utilize simple organic acid, amino acid and other small molecular substances, and meanwhile, most of other microorganisms are killed under the high-temperature condition, so that the hydrolysis process of the complex macromolecular substrate is slowed down. The hydrolysis of complex organic matter, especially lipids and proteins, in the waste is therefore the rate-limiting process affecting the efficiency of composting.
From the above, the efficiency of the current high-temperature composting technology is mainly limited to the hydrolysis link of materials, so that the fermentation efficiency cannot be greatly improved, and the composting temperature is difficult to reach higher and stable. Therefore, it is necessary to optimize the fermentation process and pretreat the materials, so that the materials are more suitable for thermophilic functional microorganisms.
Disclosure of Invention
In order to solve the technical problems, the invention provides a novel kitchen waste high-temperature composting process assisted by a complex enzyme preparation and ochrobactrum.
The complex enzyme preparation comprises the following components: laccases, i.e. polyphenol oxidases (rho-diphenol oxidases, ec1.10.3.2), belonging to the group of ceruloplasmin oxidases; alkaline esterase mix enzymes including lipase (EC 3.1.1.3), phospholipase A (EC 3.1.1.4), phospholipase B (EC 3.1.1.5); proteinase K, a serine protease with a broad cleavage activity, cleaves the carboxyl-terminal peptide bond of aliphatic and aromatic amino acids. The ochrobactrum anthropi is a novel ochrobactrum anthropi strain, is preserved in the China general microbiological culture Collection center (CGMCC) No.19230 with the preservation number of CGMCC No.19230, is named as Ochrobactumpseudointermedium by classification, is preserved in the China general microbiological culture Collection center (CGMCC No. 19230) of No. 3 of West Luo No.1 Hospital of the sunward area in Beijing within 12-23 days of 2019, and is also preserved in the CGMCC No.19230 and cultures (including subcultures), mutants and filial generations thereof, which are all called as the pseudo-intermediate ochrobactrum anthropi strain of the invention hereinafter.
The high-temperature composting process is a novel process for carrying out hydrolysis pretreatment on kitchen waste materials under the combined action of a complex enzyme and a special strain, wherein laccase, alkaline esterase, protease and ochrobactrum are used for carrying out pre-oxidation and hydrolysis treatment on crushed materials before fermentation, and heat is rapidly generated, so that the high temperature can be rapidly increased in the high-temperature composting process, the high temperature can reach more than 70 ℃ within 8 hours, and the high-temperature strain is helped to skip the hydrolysis process and directly utilize a micromolecular substrate generated by the pretreatment, so that the high-efficiency fermentation of a compost pile is realized.
The specific technical scheme of the invention is as follows: the invention provides a complex enzyme preparation and a novel kitchen waste high-temperature composting process assisted by ochrobactrum anthropi, which has the following process flow and characteristics:
(1) the water content of the kitchen waste material is required to be wide, 60-95% of the kitchen waste material can be used, the kitchen waste material is different from other common high-temperature composting processes, and the kitchen waste material does not need to be subjected to oil removal and squeezing dehydration treatment.
(2) After the materials are sorted to remove nondegradable materials such as metal, plastic and the like, the large materials are crushed by a crushing device, and the particle diameter of the crushed materials is below 10 cm.
(3) Adding the rice hull, the crushed straw, the crushed corncob or the compost clinker which accounts for 20-40% of the total mass of the materials and the CGMCC No.19230 which accounts for 1-2% of the total mass of the materials into the crushed materials, and putting the materials into fermentation equipment with a stirring function to be uniformly mixed.
(4) Adding laccase powder accounting for 1-2 per mill of the total mass of the materials into the mixed materials, and stirring for 30-90 minutes at room temperature.
(5) Adding quicklime accounting for 1-2% of the total mass of the materials into the materials after laccase treatment, fully stirring, and adjusting the pH value to 9.0-11.0.
(6) Adding lipase (EC 3.1.1.3) powder of 1-2 ‰ of total weight of the materials, phospholipase A (EC 3.1.1.4) powder of 0.2-0.8 ‰, and phospholipase B (EC 3.1.1.5) of 0.2-0.8 ‰, into the materials after pH is adjusted, and maintaining for 4-8 hr. In the process, the fertilizer pile is stirred once every 30 minutes for aeration. The fertilizer pile is quickly heated to 70 ℃ under the action of CGMCC No. 19230.
(7) The materials after temperature rise are put into high-temperature composting strains commonly used in the market, including but not limited to bacillus, Thermus, pseudomonas and other strains, and naturally generate heat and rise to 80-105 ℃ for high-temperature composting.
(8) After composting for 2-3 days, the compost pile is humified and reduced, 20-40% of clinker is discharged, and 80-60% of clinker is left in fermentation equipment.
(9) And (5) repeating the steps (1) to (6), and supplementing the fertilizer piles to the initial volume to realize semi-continuous fermentation.
The fermentation period of the traditional high-temperature compost is generally more than 7 days, otherwise the compost cannot be completely cured. The process for pretreating the materials by the complex enzyme preparation and the ochrobactrum can shorten the high-temperature composting period of 2-5 days, improve the treatment efficiency, reduce the scale of required fermentation equipment and be beneficial to the miniaturization and popularization of kitchen garbage disposal equipment.
Preferably, the reason for the cutting in (2) is that the smaller the particle size of the material, the more easily it is decomposed, but considering that while the surface area of the material is increased, a certain degree of porosity must be maintained in order to allow ventilation and to allow sufficient oxygen supply to the material, the organic material is cut and pulverized into pieces of 3 to 5cm at an optimum size here.
Preferably, the rice hull, the crushed straw, the crushed corncob or the compost clinker in the step (3) is added for adjusting the solid-liquid ratio, so that liquid accumulation in a compost pile is avoided, a complete anaerobic area is formed, and generation of malodorous substances such as sulfide and the like is possible. Therefore, the addition amount of the rice hulls, the crushed straws, the crushed corncobs or the compost clinker is based on the condition that the mixed materials have no obvious dripping phenomenon, and the excessive addition is not suitable for influencing the heat value of the compost. The optimum amount of addition is usually about 25%.
Preferably, the laccase addition amount in (4) is appropriately increased or decreased depending on the cellulose content in the compost pile. That is, when the plant waste accounts for most (namely more than half), the laccase addition amount is increased to 2 per mill; when the animal waste accounts for a large amount, the laccase addition amount can be reduced to 1 per mill.
Preferably, the pH value described in (5) is 9.5 at the most, and both esterase activity and metabolism by Ochrobactrum anthropi are considered.
Preferably, the amount of esterase to be added in (6) is appropriately increased or decreased depending on the amount of fat in the fertilizer pile. If oil separation treatment is not carried out during material collection, the maximum range value of the addition amount of various esterases in the step (6) can be selected; if the materials are subjected to oil separation treatment, the addition amount of various esterases in the step (6) can be in the minimum range.
Preferably, the ochrobactrum sp (6) may be a culture of the CGMCC No.19230 or a culture after passaging.
The invention also provides a mutant of the ochrobactrum anthropi, which is obtained by carrying out mutagenesis, domestication, gene recombination or natural mutation on the CGMCC No. 19230.
The invention also provides a bacterial culture containing the CGMCC No.19230 or the mutant.
The invention also provides a bacterial culture which is a bacterial liquid, a microbial inoculum or activated sludge and the like.
The invention also provides an enzyme preparation or protein preparation containing the CGMCC No.19230 or the mutant.
The invention also discloses the application of the CGMCC No.19230 or the mutant in sewage treatment, garbage treatment, composting, enzyme preparation production and protein preparation production.
Compared with the prior art, the invention has the beneficial effects that:
1) the strain of the invention can be metabolized and grown normally in the range of 10-80 ℃, pH 3-11 and salinity 0-5% (w/v), various monosaccharides, oligosaccharides, polysaccharides, organic acids and alcohols can be used as carbon sources, and various composite nitrogen sources such as tryptone, fish peptone, beef extract and the like can be used for growth, and the substrate spectrum is very broad.
2) The complex enzyme preparation and the bacterial strain of the invention can be used for high-temperature aerobic composting treatment of various organic matters such as kitchen garbage, agricultural product tailings, slaughterhouse wastes and the like by matching with a high-temperature composting process, so that the organic matters are completely humified, dehydrated and dried, and reduced by 70-92%, and finally the organic fertilizer with excellent quality is formed.
3) The novel high-temperature composting process also relates to special pretreatment means such as physical cutting, enzymatic hydrolysis, rapid heat generation of bacterial strain metabolism and the like, so that the compost can be rapidly heated to above 70 ℃ within 8 hours, the high-temperature stage of composting is rapidly entered, various high-temperature strains are rapidly changed into absolute dominant strains in a compost system, and on the basis of the absolute dominant strains, the continuous composting fermentation process capable of feeding and discharging materials every day is realized.
The invention is further illustrated in the following figures and detailed description. However, these drawings and specific embodiments should not be construed as limiting the scope of the invention, and modifications readily ascertainable by those skilled in the art would be included within the spirit of the invention and the scope of the appended claims.
Drawings
FIG. 1 is a colony morphology of the Ochrobactrum anthropi strain of the present invention.
FIG. 2 is a graph showing the effect of a complex enzyme on the rapid temperature rise of the ochrobactrum of the present invention.
Detailed Description
The present invention will be described in detail with reference to examples. All the experimental reagents and equipment used were, unless otherwise specified, ordinary commercially available reagents and equipment.
Unless otherwise defined, all terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs, and when partial definitions of the following terms are used, terms used in the singular may also include the plural, and vice versa. Some of the definitions of terms set forth herein are for purposes of describing particular embodiments only and are not intended to be limiting.
The term "about" means within 50%, preferably within 25%, more preferably within 10% -1% of a given value or range; or within an acceptable standard deviation of the mean.
The present invention will be further described with reference to the following examples.
Example 1 screening and identification of Ochrobactrum anthropi CGMCC No.19230
(1) Sample collection
The microorganism sample is collected from a high-temperature composting test sample of kitchen waste in a high-grade middle school dining hall in Yuhan of Yuhan district of Zhejiang province.
(2) Separation, purification and seed preservation of strain CGMCC No.19230
Self-designed, formulated "yeast sucrose YZ medium": 5g/L of yeast extract, 10g/L of sucrose, 10g/L of peptone and 5g/L of sodium chloride, and the pH value is 7.0. When the culture medium is used as a solid medium, 20g/L of agar is added.
1g of the sample is added into a conical flask filled with 100mL of sterile distilled water and a few of sterile glass beads, and the mixture is shaken for 10 minutes under the condition of 120rpm (revolutions per minute) to obtain a suspension. Diluting 1mL of suspension, coating the suspension on a sterile YZ solid culture medium, placing the suspension in a constant temperature incubator at 60 ℃ for culturing for 2-3 days, picking out a single colony after the obvious colony appears on a flat plate, repeatedly scribing and purifying, inoculating the single colony in a sterile YZ liquid culture medium, and placing the culture medium at 120rpm and 60 ℃ until the logarithmic phase. The bacterial liquid in logarithmic phase is preserved in 30% glycerin and freeze dried.
(3) Multiphase taxonomy identification of strain CGMCC No.19230
1) Phenotypic characteristics
The strains were streaked on sterile YZ solid medium and cultured at 60 ℃ for 3 days, and the colonies appeared to be soymilk-colored, about 1-2mm in diameter, convex and moist in surface, smooth in edges, round, and opaque (FIG. 1). The strain is gram-negative bacteria after gram staining.
2) Physiological and biochemical characteristics
The logarithmic phase bacterial liquid was inoculated to a sterile YZ broth at an inoculum size of 1%, and the bacterial liquid was placed at different temperatures (4 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃), different pH values (2.0, 3) of 0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0) and different NaCl salinity values (0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%), to examine the growth range of the strain. The results show that the strain can grow at 10-80 deg.C, pH 3-11, and NaCl salinity 0-5%.
Inoculating the bacterial liquid in the logarithmic phase into a culture medium with a unique carbon source (glucose, galactose, ribose, trehalose, xylose, fructose, maltose, cellobiose, starch, methanol, ethanol, sodium acetate, sodium citrate and sodium oxalate) according to the inoculation amount of 1%, placing the culture medium in an optimal culture condition, and checking the utilization condition of the bacterial strain on the carbon source. Inoculating the logarithmic phase bacterial liquid into a culture medium with a unique nitrogen source (tryptone, fish peptone, beef extract, ammonium sulfate and sodium nitrate) according to the inoculation amount of 1%, placing the culture medium in an optimal culture condition, and checking the utilization condition of the strain on the nitrogen source. The results showed that the strain could be grown using various mono-, oligo-, poly-, organic acids, alcohols as carbon sources and various complex nitrogen sources such as tryptone, fish peptone, beef extract, etc.
Inoculating the logarithmic phase bacterial liquid to a filter paper sheet with a certain specification according to the inoculation amount of 1%, placing the filter paper sheet in an optimal culture condition, and checking the degradation condition of the strain on the filter paper. Inoculating the bacterial liquid in logarithmic phase on a YZ solid culture medium containing 1g/L of carboxymethyl cellulose according to the inoculation amount of 1%, pouring 0.2% Congo red after obvious bacterial emergence, standing for 12 hours, and checking whether a transparent ring is formed on a flat plate. The results show that the strain can degrade filter paper and carboxymethyl cellulose.
Other results of biochemical experiments show that the strain has catalase activity, oxidase activity, starch hydrolase activity, casein hydrolase activity and esterase activity.
Through the identification, the strain is Ochrobactumpseudointermedium.
Example 2 fast warming Effect of Complex enzyme on Ochrobactrum
Laccase (EC 1.10.3.2) powder: food-grade laccase from Jinan Dongxuan bioengineering Co.
Lipase (EC 3.1.1.3) powder: commercially available Longda brand food grade lipase (Aspergillus oryzae, 10 ten thousand units of enzyme activity).
Phospholipase a (EC 3.1.1.4) powder: commercially available Sigma-Aldrich phospholipase A2P 6534 enzyme 9001-84-7.
Phospholipase B (EC 3.1.1.5) powder: SUNSON/SUESHEN brand lysophospholipase (food grade).
Taking 1 ton of kitchen waste, crushing the kitchen waste into fragments of about 5cm, adding 20% of dried and crushed straws, fully stirring, adding 2% of CGMCC No.19230 bacterial liquid, adding 2% of laccase powder, fully stirring, adjusting the pH value to 9.0, adding 1% of lipase (EC 3.1.1.3) powder, 0.5% of phospholipase A (EC 3.1.1.4) powder and 0.5% of phospholipase B (EC 3.1.1.5) powder, keeping for 5 hours, and stirring and ventilating once every 30 minutes. And setting a group without adding laccase and alkaline esterase powder as a control group, and detecting the temperature every 1 hour to check the influence of the compound enzyme on the temperature rise of the strains. The result shows (figure 2), the complex enzyme group can promote the fast temperature rise of the ochrobactrum anthropi, the compost can reach 70 ℃ within 8 hours, and the temperature can be maintained at 70-80 ℃.
Example 3: complex enzyme preparation and application of ochrobactrum anthropi in high-temperature composting
1: composting treatment of kitchen waste
2 tons of kitchen garbage are taken, and the solid-liquid mixture is cut and stirred to change the solid into fragments with the diameter of about 5 cm. The solid-liquid garbage is put into a fermentation device with a stirring function, 600Kg of dried rice hulls are added, and the mixture is fully mixed and stirred uniformly. 20Kg of CGMCC No.19230 was added, and 3Kg of laccase powder was added and stirred at once per minute for 60 minutes. Adding 40Kg of raw stone ash into a fertilizer pile for multiple times, uniformly stirring to ensure that the pH value reaches 9.5, adding 5Kg of esterase mixture, and uniformly stirring. The compost heap was then aerated with stirring once every 30 minutes and after a 6 hour hold the temperature was raised to 70 ℃. And (3) adding heat-resistant bacillus to perform high-temperature composting, maintaining the temperature between 70 and 100 ℃, completing composting fermentation after 2 days, and reducing the weight of the materials by 70 percent. After discharging half of the clinker, replacing half of the dry rice hulls with the remaining clinker to replenish 1.8 tons of kitchen garbage again, and repeating the steps to realize semi-continuous fermentation.
2: compost of slaughter waste
1 ton of slaughter house fertilizer is cut and stirred to change the solid into pieces with the diameter of about 3 cm. And putting the solid-liquid garbage into a fermentation device with a stirring function, adding 30% of dried straw crushed materials, and fully mixing and stirring uniformly. 10Kg of CGMCC No.19230 was added, and 1Kg of laccase powder was added and stirred at a frequency of once per minute for 60 minutes. Adding 5Kg of raw stone ash into the fertilizer pile for multiple times, stirring uniformly to make the pH value reach 9.5, adding 2Kg of esterase mixture, and stirring uniformly. The compost heap was then aerated with stirring once every 30 minutes and after 8 hours the temperature was raised to 70 ℃. And (3) putting commercial EM microbial inoculum for high-temperature composting, maintaining the temperature between 70 and 80 ℃, and completing composting fermentation after 3 days.
3: compost of agricultural and sideline products
10 tons of straws are taken, added into a fermentation device with cutting and stirring functions to be continuously cut and stirred into fragments of about 6cm, 100Kg of CGMCC No.19230 is put into the fermentation device, 20Kg of laccase powder is added, and the mixture is stirred for 60 minutes at the frequency of once per minute. 80Kg of raw stone ash was added to the fertilizer pile several times, stirred uniformly until the pH reached 9.5, and 1Kg of esterase mixture was added, stirred uniformly. The compost heap was then aerated with stirring every 30 minutes and after 4 hours the temperature was raised to 70 ℃. And (3) putting a commercially available straw composting microbial inoculum for high-temperature composting, maintaining the temperature between 80 and 95 ℃, and completing composting fermentation after 4 days.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
While the invention has been described in detail with respect to the preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing and various other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention.

Claims (8)

1. The Ochrobactrum sp, which is characterized by being preserved in China general microbiological culture Collection center (CGMCC) at 23.12.2019 with the preservation number of CGMCC No.19230 and is named as Ochrobactrum sp.
2. A culture or a passaged culture of the Ochrobactrum anthropi according to claim 1.
3. The mutant of ochrobactrum of claim 1, wherein the mutant is obtained by subjecting the ochrobactrum of claim 1 to mutagenesis, domestication, genetic recombination or natural mutation.
4. A bacterial culture comprising the ochrobactrum of claim 1 or comprising the mutant of claim 3.
5. A mixture comprising the bacterial culture of claim 4, wherein said mixture is a bacterial solution, a bacterial agent, or an activated sludge.
6. An enzyme preparation or protein preparation comprising the ochrobactrum of claim 1 or comprising the mutant of claim 3.
7. Use of the ochrobactrum of claim 1 or the mutant of claim 3 for sewage treatment, waste treatment, composting, production of enzyme preparations, production of protein preparations, wherein the composting is preferably a high temperature composting fermentation.
8. A high-temperature composting fermentation method using the ochrobactrum of claim 1 or the mutant of claim 3, characterized by comprising the steps of:
(1) collecting kitchen waste materials, wherein the water content of the kitchen waste materials is about 60% -95% of the total materials;
(2) sorting the kitchen waste materials in the step (1), removing non-degradable substances, and crushing until the particle diameter of the materials is less than 10cm, preferably 3-5cm, wherein the non-degradable substances are preferably metal, plastic and the like;
(3) adding rice hulls, crushed straws, crushed corncobs or compost clinker accounting for about 20-40%, preferably 25%, of the total mass of the crushed materials and ochrobactrum anthropi according to claim 1 or the mutant according to claim 3 accounting for about 1-2% of the total mass of the crushed materials into the crushed materials obtained in the step (2), and then uniformly mixing, wherein the ochrobactrum anthropi according to claim 1 or the mutant according to claim 3 is preferably in a liquid state, and the uniformly mixing step is preferably carried out in a fermentation device with a stirring function;
(4) adding laccase powder accounting for 1-2 per mill of the total mass of the mixture into the mixture obtained in the step (3), and stirring for about 30-90 minutes at room temperature, wherein preferably, when the plant waste accounts for more than half of the mass of the mixture, the addition amount of the laccase is 2 per mill of the total mass of the mixture; when the animal waste accounts for more than half of the total mass of the mixed material, the addition amount of the laccase is 1 per mill of the total mass of the mixed material;
(5) adding quicklime accounting for 1-2% of the total mass of the laccase-treated material into the laccase-treated material obtained in the step (4), fully stirring, and adjusting the pH value to about 9.0-11.0, preferably 9.5;
(6) adding lipase (EC 3.1.1.3) powder accounting for about 1-2 per thousand of the total mass of the material after the pH is adjusted, phospholipase A (EC 3.1.1.4) powder accounting for about 0.2-0.8 per thousand of the total mass of the material after the pH is adjusted, and phospholipase B (EC 3.1.1.5) powder accounting for about 0.2-0.8 per thousand of the total mass of the material after the pH is adjusted, keeping for 4-8 hours, stirring the material once every 30 minutes for aeration until the temperature of the material is raised to about 70 ℃, and preferably, if oil separation treatment is not carried out during the collection of the material, measuring the maximum value of the enzyme addition amount range; if the material is subjected to oil separation treatment, the enzyme addition amount can be in the minimum range;
(7) adding high-temperature composting strains commonly used in the market into the heated material obtained in the step (6), wherein the high-temperature composting strains preferably include but are not limited to bacillus, Thermus, pseudomonas and other strains, and heating the material to 80-105 ℃;
(8) completing humification and reduction after composting the materials obtained in the step (6) for 2-3 days, discharging 20-40% of clinker, and leaving 80-60% of clinker in fermentation equipment;
(9) and (5) repeating the steps (1) to (6), and supplementing the fertilizer piles to the initial volume to realize semi-continuous fermentation.
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